1. Field of the Invention
The invention pertains to manufacturing robust devices employing particle plasmon resonance to improve microarray bioassay performance.
2. Description of Related Art Including Information Disclosed Under 37 CFR 1.97 and 37 CFR 1.98.
The use of metallic nanostructures to create altered spectral effects from fluorescent molecules has been known for decades. Both the fluorescent intensity and lifetime can be beneficially altered using metal nanostructures. The most relevant references, e.g., U.S. Pat. No. 5,866,433 ('433) and U.S. Pat. No. 5,837,552 ('552), both incorporated herein by reference, describe embodiments that in one manner or another place metal nanoparticles on substrates and use specific biological binding to draw fluorescent material into the vicinity of the metal nanoparticle layer.
The prior art has described a substrate, which can effectively be any supporting structure, planar or three-dimensional, upon which a layer of conductive nanoparticles are made to adhere. Nanoparticles are generally objects less than 1 micron in lateral and axial dimensions, and for applications to fluorescent enhancement are generally described as smaller than a wavelength of the “light”. The reference to “light” to in the prior art is often vague. Since this is a fluorescent system, there are at least two spectra to consider, the excitation and fluorescent emission spectra. Since, in general these partially overlap and have peaks fairly close to each other, the distinction between which wavelength is more important has not been clarified in the prior art. There is however, a decisive physical phenomenon that clearly dictates the size of nanoparticles for optimum enhancement, that being nanoparticle plasmon resonance.
The “size” of nanoparticles is also vague in the prior art. Size and shape are in fact both important in the enhancement mechanisms and some recent literature has begun to explore the importance of nanoparticle shape. In the conventions of the prior art, if a nanoparticle is a flat disk, the size would likely refer to its lateral dimension defined by the diameter. If the nanoparticle were rod shaped, the size might be taken as the length, however, the diameter of the rod is also important. The details of size and shape that accrue to optimized enhancement are not completely understood, so there is a necessary void in the descriptions found in prior art. Therefore the prior art provides only clues regarding the ability to reliably and repeatedly fabricate nanoparticles with predictable behaviors.
The thickness of nanoparticles, optical properties of the nanoparticle film, spatial density of the nanoparticles within the film, as well as the number of layers of nanoparticles comprising the “film” are all referred to generally in the prior art. It is taken by most references and specifically by '433 and '552 that the nanoparticles should have a thickness in the range of 2 nm to 25 nm. Nevertheless, vacuum and chemical deposition methods are capable of extremely well defined layer thickness, and the need for generality is unclear. In fact, some references teach metal islands spaced apart, or metal islands to spaced apart that may be connected by thinner or different metal structures, or metal islands that may be touching. In each of these cases it can be successfully shown that an enhancing effect occurs, however, the physical mechanisms and design parameters required to provide a maximum effect are not taught. This is largely because the design of an enhancing structure is not just a nanoparticle design problem, it is a system design problem.
Another interesting factor emerges when considering the nature of nanoparticles described in the prior art. Since the very first experiments were conducted over 25 years ago using chemically deposited silver, the method of chemical deposition has been observed and repeated by others. This method does result in a layer of island-like discrete nanoparticles that, in general do not contract each other. It is also possible to obtain such “metal island” films using vacuum deposition methods such as sputtering or thermal evaporation. These films are often semi transparent to incident light since the metal film has a relatively low spatial density. These metal island films are described in all of the prior art. It has been found, however, that thin, conductive, solid films of many metals can be deposited with high adhesion and high fluorescence enhancement, however, these films are not comprised of island-like metal structures. They are instead comprised of columnar metal crystals typically expected by those skilled in the art of thin film vacuum deposition. These metal layers, usually being well over 20 nanometers thick, are characterized by a columnar nanostructure that has surfaces that “appear” to be bumpy. The bumps have the appearance of nanoparticles when viewed using an atomic force microscope (AFM) or scanning electron microscope (SEM), however, they are not discrete particles and behave electrically differently than the discrete particle films of prior art. Interestingly, if the surface features, bumps, have apparent diameters in the range of 20 to 300 nm fluorescence enhancement is observed similar to that seen with island films. The physical phenomena accounting for the coupling of electrical field energy from an exciting light field to the plasmon field of this nanostructured surface is not well defined in theory, but it is clear that the metallic structures are completely different from the island-like structures found in the prior art. Further, it is found that the conditions for optimum fluorescence enhancement are also markedly different from those required for island structures. This invention addresses enhancement mechanisms created using thin, deliberately nanostructured, continuous metal films that are not made of island-like metal nanoparticles. Therefore, there is a clear distinction between the terms nanoparticle and nanostructure as used herein. Much of the background discussion relating to the prior art of island-like nanoparticle films is nevertheless relevant. In this discussion the terminology of the prior art is used where appropriate and will be distinguished from methods of the invention using nanostructured films.
It is well recognized in the prior art that some form of preparation must be performed on the substrate supporting the enhancing metallic nanoparticles. Silanization is taught in '552, and other methods of causing metals to adhere to plastics and glasses are well known in industry. Nevertheless, the method of adhering the active metal to a substrate can interact with the operative physical mechanisms and cause the resulting enhancement to be greater or smaller. Further, in some structures, the adhesion process can be vulnerable to external chemical attack leading to definite limitations in the lifetime and practicality of a product. Similarly, metalization layers used to create adhesion, e.g., chromium, tungsten, titanium, palladium and others can alloy with the active nanoparticles and change their plasmonic properties; resonance, damping, etc. Moreover, a design that has all dimensions optimized for one set of materials will not be optimum for a different choice of materials. The prior art alludes only vaguely to the attachment methods, yet, without adhesion between the active nanoparticles and the substrate, a practical structure cannot be built. While the need for adhesion may seem obvious, the proper choice of materials and processes that results in a well adhering film with substantial enhancement is indeed not obvious and is not taught elsewhere.
The enhancement mechanism is known to be due to the mutual coupling of energy from a plasmon electrical field to the fluorophore. It is well known and taught that nanoparticle and surface plasmon energetic coupling mechanisms, like all energetic coupling mechanisms, has distance dependence. In fact, in general, it has more than one distance dependent mechanism, is highly non-linear, as has only recently been partially understood. The methods and parameters enabling the ability to design reliably working structures that make deliberate use of the distance dependencies have not been taught. As measured from the original surface of the substrate, the prior art speaks of accomplishing the required separation between the mean surface height of the metal nanoparticle layer, and the mean fluorophore location distance by using an intervening layer of material which may or may not be part of the biological assay system. Layers are called biorecognitive or coupling layers and have been exemplified as layers of tissues, polymers, or other materials. In fact, all of these are completely viable methods, as is also, for example, choosing an appropriate DNA strand length which binds a labeled DNA analyte in a manner to place the fluorescent labeling molecule an appropriate distance from the metal surface. However, the term “appropriate distance” is unclear in the prior art. The prior art does not reveal that if one defines the system of materials used in a specific enhancing structure, the optimum separation distance can be specified empirically with reliable precision leading to optimized, manufacturable and reliable structures.
It is well taught that microarray substrates are exposed to many harsh compounds, salts, acids, and bases, often at elevated temperatures. Metallic films, such as silver, are subject to corrosion. Much work through the ages has gone into finding coatings that can protect mirrors and other optical surfaces. In the case of optical instruments, the films requiring protection are generally many microns thick and are solid uniform films. In comparison, the art of nanoparticle plasmon devices requires films of nanoparticles, or nanoclusters, of metal. Working films are usually less than 50 nanometers thick and are made of tiny grains of material, all of which are particularly vulnerable to chemical attack. After hundreds of attempts by the inventors and others to fabricate a robust, chemically resistant film of silver nanoparticles, it appears that the prior art has overlooked or underestimated the difficulty of using such films in real, practical biotechnology applications. Devices built according to the descriptions available in all literature discovered by this team failed almost immediately when exposed to assay protocols. So, while the science revealed elsewhere is of great fundamental value, the practical application of nanoparticle plasmon technology to microarrays has not been previously accomplished.
It is broadly recognized throughout the entire community of microarray and microplate users, including both researchers and applied clinicians that the data quality of microarrays needs to be improved in the interest of more rapidly advancing to human health. Critical needs exist in cancer research, viral research, and drug discovery for more sensitive and accurate means of discovering disease and the cures for disease. As regards the specific problems facing microarray users, few truly effective methods have been found to accomplish this illusive goal. The use of metal enhanced fluorescence has been recognized by a few to offer a potential solution. Yet, while the prior art successfully characterizes a general phenomenon and indicates a broad range of parameters for fabricating devices, the prior art fails to address most of the critical parameters with sufficient detail or accuracy to permit the fabrication of practical structures. The prior art frequently and eloquently describes bioassay principles and well known bioassay surface preparation methods, yet none describe a practical manufacturing protocol for a nanoparticle plasmon sensor embodiment that absolutely improves assay sensitivity in a commercially viable embodiment. Summarizing the prior art it is clear that a need exists for a microarray substrate comprising a material system that provides: stable material adhesion to glass and plastic substrates; high plasmon field enhancement; high coupling of enhanced plasmon energy into fluorescent molecules causing significantly enhanced fluorescence emission; and simultaneously being substantially free from corrosion in bioassay buffer solutions. The prior art has focused on the use of discrete nanoparticle films which have distinct behaviors and limitations. The improved invention of this application addresses these issues and results in a complete description of several practical and practicable embodiments of assay substrates of significant value to the life sciences.
Publications in the technical area.
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